4-aminoalkylamino-2-halobenzyl alcohols and alkyl ethers thereof

ABSTRACT

4-8AMINO-(POLYCARBON-LOWER-ALKKYL)-AMINO) - 2 - HALOBENZYL ALCOHOLS AND LOWER-ALKYL ETHERS THEREOF HAVE SCHISTOSOMACIDAL ACTIVITY. THE BENZYL ALCOHOLS ARE PREJPARED: BY RREDUCTION OF THE CORRESPONDING BENZALDEHYDES OR LOWER-ALKYL BENZOATES; BY MICROBOILOGICAL OXIDATION OF THE CORRESPONDING 4-SUBSTITUTED-AMINO-2-HALOTOLUENES; OR, FOR THE COMPOUNDS WHERE POLYCARON-LOWER-ALKYL IS ETHYLENE, BY REDUCING THE CORRESPONDING LOWER-ALKYL 4-(AMINOACETYLAMINO)-2-HALOBENZOATES OR BY REDUCING THE CORRESPONDING 4 - (AMINOACETYLAMINO)-2-HALOBENZYL ALCOHOLS. THE ETHERS ARE PREPARED BY THE HEATING THE BENZYL ALCOHOLS WITH A LOWER-ALKANOL IN THE PRESENCE OF AN ACID.

United States Patent 3,714,167 4-AMINOALKYLAMINO-2-HALOBENZYL ALCO- HOLS AND ALKYL ETHERS THEREOF Sydney Archer, Bethlehem, and David Rosi, East Greenbush, N.Y., assignors to Sterling Drug Inc., New

York, N.Y.

N0 Drawing. Continuation-impart of application Ser. No. 664,628, Aug. 31, 1967, now Patent No. 3,558,629, dated Jan. 26, 1971, which is a continuation-in-part of application Ser. No. 444,848, Apr. 1, 1965, now Patent No. 3,379,620, dated Apr. 23, 1968. This application June 2, 1970, Ser. No. 42,876

Int. Cl. C07d 51/70 US. Cl. 260-268 C 11 Claims ABSTRACT OF THE DISCLOSURE 4-[arnino-(polycarbon-lower-alkyl)-arnino] 2 halobenzyl alcohols and lower-alkyl ethers thereof have schistosomacidal activity. The benzyl alcohols are prepared: by reduction of the corresponding benzaldehydes or lower-alkyl benzoates; by microbiological oxidation of the corresponding 4-substituted-amino-2-halotoluenes; or, for the compounds where polycarbon-lower-alkyl is ethylene, by reducing the corresponding lower-alkyl 4-(aminoacetylarnino)-2-halobenzoates or by reducing the corresponding 4 (aminoacetylamino)-2-halobenzyl alcohols. The ethers are prepared by heating the benzyl alcohols with a lower-alkanol in the presence of an acid.

This application is a continuation-in-part of our copending application Ser. No. 664,628, filed Aug. 31, 1967 and now US. Pat. 3,558,629, issued J an. 26, 1971, which in turn is a continuation-in-part of our application Ser. No. 444,848, filed Apr. 1, 1965, now US. Pat. 3,379,620, issued Apr. 23, 1968.

This invention relates to 4-aminobenzyl alcohol derivatives and to processes for their preparation.

The invention sought to be patented, in its composition aspect, resides in the class of compounds which we designate 4 [amino-(polycarbon-lower-alkyl)amino]-2-halobenzyl alcohols and lower-alkyl ethers thereof. The tangible embodiments of this composition aspect of the invention possess the inherent applied use characteristics of having schistosomacidal properties, as determined by standard chemotherapeutic evaluation procedures.

Said application Ser. No. 444,848 now US. Pat. 3,379,- 620 discloses and claims the process for producing a 4- [amino-(polycarbon-lower-alkyl)amino] 2 halobenzyl alcohol which comprises subjecting a 4-[amino-(polycarbon-lower-alkyl)-amino]-2-halotoluene to the fermentative enzymatic action of an organism capable of effecting oxidation of the l-methyl group to l-hydroxymethyl, said organism classified in the orders Moniliales, Mucorales, Sphaeriaeles, Sphaeropsidales, Melanconiales and Actinomycetales. We have found variations exist in the efficiency of different genera and species of organisms within the above orders in our oxidative process and have therefore ascertained that, by following the screening procedure hereinafter described, the eifectiveness of any particular organism can readily be determined.

Another process of preparing said 4-[amino-(polycarbon-lower-alkyl)-amino)-amino] 2 halobenzyl alcohol comprises reacting a 4-[amino-(polycarbon-lower-alkyl)- amino] 2 halobenzaldehyde or lower-alkyl 4- [amino- (polycarbon-lower-alkyl)-amino]-2-halobenzoate with a reducing agent effective to reduce, respectively, benzaldehydes or lower-alkyl benzoates to benzyl alcohols.

Our copending application Ser. No. 664,628, now US. Pat. 3,558,629, discloses and claims the intermediate 4- (l-piperazinyl)-2-halobenzaldehydes which are prepared by reacting a 3 (1 piperazinyl)-halobenzene with an N'R'-N-R"-formamide, where R' is lower-alkyl and R" is lower-alkyl or phenyl, in the presence of phosphorus oxyhalide.

Well known as schistosomacidal agents are a wide variety of 4 [amino-(polycarbon-lower-alkyl)-amino]- 2-halotoluenes which differ from our 4- [amino-(polycarbon-lower-alkyl)-amino]-2-halobenzyl alcohols or ethers in having methyl instead of hydroxymethyl or alkoxymethyl as a substituent on the benzene ring ortho to halo and para to the aminoalkylamino substituent. The amino- (polycarbon-lower-alkyl)amino substituent of these known 4 [amino (polycarbon-lower-alkyl)amino]-2- halotoluenes varies widely; for example, in some compounds the t-wo amino groups are connected by alkylene having a chain of two to six carbon atoms inclusive, optionally substituted by lower-alkyl, and having its connecting linkages on different carbon atoms, with the terminal amino being primary, secondary or tertiary, and the other amino (directly connected to the benzene ring) bearing hydrogen or lower-alkyl; in other compounds the two amino groups are part of a piperazino ring, with the terminal amino (i.e., the 4-ring nitrogen of piperazino) being unsubstituted or bearing various substituents, e.g., lower-alkyl, lower-alkenyl, lower-hydroxyalkyl, phenyl- (lower-alkyl), carbamyl, thiocarbamyl, loWer-carbalkoxy, carboxy- (lower-alkanoyl), carboxy-(lower-alkenoyl), etc. Also, these compounds can bear other low-molecular substituents, e.g., lower-alkyl, at one or more of the remaining three available ring positions, i.e., 3, 5 and/orb. One of the best known and most active of these 4-(aminoalkylamino) 2 halotoluenes is 2-chloro-4-(2-diethylaminoethylamino)toluene, preferably named 3-chloro-N- (Z-diethylaminoethyl)-4-methylaniline and perhaps better known as ,Mirasam other highly active and preferred members of this known class of compounds are 1-(3- chloro-4-methylphenyl)piperazine [same as 2 chloro-4- (1-piperazinyl)toluene], 1-(3-chloro 4 methylphenyl)- 4-methylpiperazine, maleic acid mono 4 (3-chloro-4- methyl)piperazide and 2-chloro-4-{4-[6-(4-tertiary-amylphenoxy)hexyl]-1-piperazinyl}-toluene. All of these and other heretofore known compounds having high schistosomacidal activity required the presence of the methyl substituent on the benzene ring, as well as the halo and amino-(polycarbon-lower-alkyl) amino substituents at ortho and para positions, respectively. Workers in the field have reported that the methyl group cannot be replaced by another radical without losing the schistosomacidal activity.

We have now found that compounds of this type where the ring methyl substituent is replaced by hydroxymethyl, i.e., 4 [amino-(polycarbon-lower-alkyl)amino]-2halobenzyl alcohols, or by lower-alkoxymethyl not only have high schistosomacidal activity but also are more active as schistosomacidal agents in hamsters and less toxic than the corresponding methyl compounds.

Without limiting the generality of the foregoing, illustrative and preferred embodiments of our 4-[amino- (polycarbon-lower-alkyl)amino] 2 halobenzyl alcohols and lower-alkyl ethers are those of Formula I CHzOR hal R 1&3

where ha] is halo, i.e., chloro, bromo, iodo or fiuoro; R R and R are each hydrogen or lower-alkyl; NB is R is hydrogen or lower-alkyl; Y is polycarbon-lower-alkylene; R and R are each hydrogen or lower-alkyl, loweralkenyl or lower-hydroxyalkyl and can be the same or different; R and R taken with N also comprehend saturated N-heteromonocyclic radicals having from five to six g atoms, illustrated by piperidino, pyrrolidino, morpholino, piperazino, hexamethyleneimino and lower-alkylated derivatives thereof; and, R is hydrogen, lower-alkyl, loweralkenyl, lower-hydroxyalkyl, carbamyl, thiocarbamyl, lower-alkanoyl, lower-carbalkoxy, carboxy-(lower-alkanoyl), carboxy-(lower-alkenoyl) or phenyl-X- (loweralkyl), where X is oxygen or a direct linkage. Here and elsewhere throughout this specification, it will be understood the benzene ring or phenyl can bear any member and kind of substituents such as would occur to the man skilled organic chemistry, e.g., such substituents, solely for illustration and without limitation, including loweralkyl, lower-alkoxy, halo (chloro, bromo, iodo or fluro), nitro, lower-alkyl-mercapto, lower-alkanoylamino, loweralkanoyloxy, lower-alkylamino, lower-alkenyl, and the like.

The compounds of Formula I where NB is are disclosed and claimed in our copending application Ser. No. 221,047, filed Jan. 26, 1972.

The above terms, as used throughout this specification, have the following meanings, each of which are illustrated but without limiting their generality: lower-alkyl means alkyl radicals having from one to six carbon atoms which can be arranged as straight or branched chains, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl and n-hexyl; polycarbon-lower-alkylene" means alkylene radicals having from two to six carbon atoms inclusive and having its connecting linkages on different carbon atoms, e.g., CH CH -CH(CH )CH 'CH2CH2CH2CH2, 5- and -(CH2)6 lower-alkenyl means alkenyl radicals having from three to six carbon atoms, e.g., 2-propenyl (allyl), 2-methyl-2- propenyl, Z-butenyl, S-butenyl and 2-hexenyl; lower-hydroxyalkyl means hydroxyalkyl radicals having from two to six carbon atoms and having its connecting linkage and the hydroxyl group on different carbon atoms, e.g., Z-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxy-2-methylpropyl, 4-hydroxybutyl, 2-hydroxy- 2,2-dimethylpropyl and 6-hydroxyhexyl; lower-carbalkoxy means carbalkoxy radicals where the alkoxy portion can be straightor branch-chained and has from one to six carbon atoms, e.g., carbomethoxy, carbethoxy, carbo-n-propoxy, carbisopropoxy, carbo-n-butoxy and carbo-n-hexoxy; lower-alkanoyl means alkanoyl radicals having from one to six carbon atoms which can be arranged as straight or branched chains, e.g., formyl, acetyl, propionyl (n-propanoyl), isobutyryl (Z-methyl-n-propanoyl) and caproyl (n-hexanoyl); lower-alkenoyl means alkenoyl radicals having from three to six carbon atoms, e.g., 2-propenoyl, 2-methyl-2-propenoyl, 2-butenoyl, 3-

v butenoyl and 2-hexenoyl. When NR R of Formula I or n-propylpiperidino, 2,2-dimethylpiperidino, 2-methylpyrrolidino, 2,5-dimethylpyrrolidino, 2,3-dimethylmorpholino, 2-ethylmorpholino, Z-methylhcxamethyleneimino, 2,7-dimethylhexamethyleneimino, 4-methylpiperazino, 3-ethylpiperazino, 2,4,6-trimethylpiperazino, and the like.

The 4 [amino-(polycarbon-lower-alkyl)-amino] 2- halobenzyl alcohols and ethers of our invention are useful in the free base form or in the form of their acid-addition salts, and both forms are within the purview of the invention, and are considered to be one and the same invention. The acid-addition salts are simply a more convenient form for use; and, in practice, use of the salt form inherently amounts to use of the base form. The acids which can be used to prepare the acid-addition salts are preferably those which produce, when combined with the free base, chemotherapeutically acceptable salts, that is, salts whose anions are relatively innocuous to the animal organism in chemotherapeutic doses of the salts, so that the beneficial properties inherent in the free base are not vitiated by side effects ascribable to the anions; in other words, the latter do not substantially affect the chemotherapeutic properties inherent in the cations. In practicing our invention, we found it convenient to employ the hydrochloride or p-toluenesulfonate salt. However, other appropriate chemotherapeutically acceptable salts within the scope of the invention are those derived from mineral acids such as hydrobromic acid, hydriodic acid, nitric acid, phosphoric acid, sulfamic acid, and sulfuric acid; and 01'- ganic acids such as acetic acid, citric acid, tartaric acid, lactic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, quinic acid, 3-hydroxy-2-naphthoic acid, pamoic acid (2,2'-dihydroxy-1,l'-dinaphthylmethane-3,3-dicarboxylic acid), naponic acid 1,5-naphthalenedisulfonic acid) and the like, giving the hydrobromide, hydriodide, nitrate, phosphate, sulfamate, sulfate, acetate, citrate, tartarte, lactate, methanesulfonate, ethancsulfonate, quinate, 3-hydroxy-2-naphthoate, pamoate and naponate, respectively.

The acid-addition salts are prepared preferably by reacting the free base and acid in an organic solvent, e.g., ethanol, acetone, etc., in which case the salt separates irectly or can be obtained by concentration of the solution.

Although chemotherapeutically acceptable salts are preferred, all acid-addition salts are within the scope of ur invention. All acid-solution salts are useful as sources f the free base form even if the particular salt per se is not desired as the final product, as for example when the sa t is formed for purposes of purification or identification, or when it is used as an intermediate in preparing a chemotiherapeutically acceptable salt by ion exchange proceures.

Also encompassed by our invention are simple esters of our 4 [amino-(polycarbon-lower-alkyl)-amino]-2-halobenzyl alcohols, for example, lower-alkanoates (e.g., acetate, propionate and butyrate), phenylcarboxylates (e.g., benzoate, 3,5-dinitrobenzoate and 4-ethoxybenzoate) and carbanilates. These esters are prepared by known means and are useful to further characterize and identify 0 1" substituted-benzyl alcohols.

The molecular structures of the 4-[amino-(polycarbonlower-alkyl)amino]-2-halobenzyl alcohols of our invention were assigned on the basis of evidence provided by infrared, ultraviolet and nuclear magnetic resonance spectra, by chromatograph mobilities, and, by the correspondence of calculated and found values for the elementary analyses for representative examples.

The manner of making and using our invention will now be generally described so as to enable the person skilled in the art of microbiological or organic chemistry to make and use the same.

Final products from corresponding benzaldehydes or lower-alkyl benzoates This process utilizes as intermediates 4-[amino-(polycarbon-lower-alkyl) amino] 2 halobenzaldehydes or lower-alkyl 4 [amino-(polycarbon-lower-alkyl)-amino]- 2-halobenzoates, which are prepared by generally known procedures illustrated in the specific exemplary disclosure below or, in the case of the 4-aminoalkylamino-Z-halobenzaldehydes, by another process hereinbelow described. The process comprises reacting the 4-aminoalkylamino-2- halobenzaldehyde or lower-alkyl 4-aminoalkylamino-2- halobenzoate with a reducing agent effective to reduce benzaldehydes or lower-alkyl benzoates, respectively, to benzyl alcohols. The reduction can be carried out both by catalytic hydrogenation and by chemical methods. Catalysts suitable when catalytic hydrogenation is used include Raney nickel, platinum or palladium. Suitable chemical reducing agents include: alkali aluminum hydrides, e.g., lithium aluminum hydride; alkali borohydrides, e.g., sodium borohydride; an alkali metal and a lower alkanol, e.g., sodium and ethanol. Also, the benzaldehydes can be reduced to the benzyl alcohols using iron and acetic acid or isopropyl alcohol and aluminum isopropoxide. In practicing our invention, we preferably used lithium aluminum hydride in ether or tetrahydrofuran in reducing the benzaldehydes and sodium borohydride in a lower alkanol, e.g., ethanol or methanol, with or without water, in reducing the lower-alkyl benzoates.

A modification of this process comprises reacting a lower-alkyl 4-aminoacetylamino-2-halobenzoate, e.g., without limiting the generality of the foregoing, a lower-alkyl 4-aminoacetylamino-2-halobenzoate of the Formula II ('3 O O-(lower-alkyl) hal where hal, R, R R R and R have the meanings given above for Formula I, with a reducing agent effective to reduce lower-alkyl benzoates to benzyl alcohols and aminoacetamides to aminoethylamines. Suitable reducing agents are those given above for the reduction of loweralkyl benzoates to benzyl alochols. We preferably used an alkali aluminum hydride, e.g., lithium aluminum hydride in an inert solvent, e.g., tetrahydrofuran, in the presence of a suitable catalyst, e.g., aluminum trichloride; best results were obtained using lithium aluminum hydride, tetrahydrofuran and aluminum chloride, and keeping the reaction temperature below 0? C., preferably between C. and 5 C. Alternatively, the same reduction can be carried out but using instead of the benzoates of Formula II the corresponding benzyl alcohols, i.e., CH OH in place of COO-(lower-alkyl). These intermediate 4-aminoacetylamino-Z-halobenzyl alcohols, which also are useful as local anesthetic agents, as determined by standard pharmacological evaluation procedures, can be prepared from the corresponding 4-aminoacetylamino-Z-halotoluenes by using our microbiological oxidative procedure described hereinabove. The intermediate 4-aminoacetylamino-Z-halotoluenes are conveniently prepared in two generally known steps from the corresponding known 4-amino-2-halotoluenes, that is, by first reacting a 4-amino-2-halotoluene with chloroacetyl chloride to form a 4-(chloroacetylamino)-2-halotoluene which is then reacted with ammonia or appropriate amine of the formula HNR R (R and R defined above) to yield the 4-(R R N-acetylamino)-2-halotoluene.

Formylation of 3-(l-piperazinyl)halobenzenes This process comprises reacting a 3-(1-piperazinyl)- halobenzene with an N-RN -formamide, where R' is lower-alkyl and R" is lower-alkyl or phenyl, in the presence of phosphorus oxyhalide to form the correspondprepared by heating the benzyl alcohol (Formula I where R is hydrogen) with the appropriate lower-alkanol in the presence of an acid, preferably a strong acid. This reaction is conveniently carried out heating the basic benzyl alcohol as its acid-addition salt, e.g., di(p-toluenesulfonate), in the lower alkanol at its boiling point to dissolve the benzyl alcohol salt and allowing the resulting solution to cool whereupon the basic lower-alkyl ether separates as its acid-addition salt.

The foregoing discussion is offered to illustrate the various aspects of our invention and not to limit its scope. Our invention is further illustrated by the following examples.

EXAMPLE 1 Fermentative enzymatic oxidation of 2-chloro-4-(lpiperazinyl)toluene to 2-chloro-4-(l-piperazinyl)-benzyl alcohol was accomplished as follows: Four 10 liter fermentations employing Aspergillus sclerotiorum (SWRI A available at Sterling-Winthrop Research Institute, Rensselaer, N.Y.) in sterile soy-dextrose medium of the following composition were carried out.

Dextrose 1000 Soybean meal Yeast 50 NaCl 50 MgSO .nH O 2.5 NZiHgPO4-H2O 1 8 NE2HPO4- Tap water, 9.5 liters.

pH adjusted to 7.3 with 10 N HCl; autoclave at 120 C.

and 15 p.s.i. for 15 minutes.

The stock culture was initially grown at 26 C. on slants in a nutrient medium (e.g., maltose, 4-0' g./liter, and Proteose Peptone No. 3, 10 g./liter) in 22 x mm. tubes for ten to fourteen days. These slants were used to prepare seed for the l0-liter fermenters as follows: Sterile distilled water (10- ml.) was added to a slant and the spores and some vegetation growth were scrapped with a sterile hooked needle. The resulting suspension was added to a two liter flask containing 700 ml. of sterile soy-dextrose medium of the following composition:

G. Soybean meal l5 Dextrose 20 Yeast 5 NaCl 5 K HPO 5 Tap water, 1 liter.

pH adjusted to 6.4 with 10 N HCl prior to autoclaving at 121 C. and 1S p.s.i. for 15 minutes.

These cotton plugged flasks were incubated at 26 C. for approximately seventy hours on a rotary shaker with a one-inch throw at 240 rpm. Each flask provided seed for one 10-liter fermenter. The fermentations were carried out at 28 C. in a water bath with an air flow of 5 liters per minute and were agitated at 450 rpm. After an initial twenty-four hours growth, the substrate, 2-chloro- 4-(l-piperazinyl)toluene as its hydrochloride salt, 212 g. (53 g. to each fermenter), was added in portions (5-40 g.) over a period of five days. The tanks were assayed for 2-chloro-4-(l-piperazinyDbenzyl alcohol content periodically as follows: Samples from each fermenter were made basic with 0.2 ml. of 10 N sodium hydroxide solution and extracted with methylene dichloride. The extracts were evaporated to dryness and the residues dissolved in 3 ml. of methylene dichloride. Aliquots (20 ,ul.) were transferred to thin-layer silica gel plates impregnated with 1% of a phosphor (e.g., Radelin GS-115). The plates were developed in a solvent containing 8: 1 :1 parts by volume of OH Cl :CH OH:*N(C -H The components were viewed as blue spots on a yelow fluorescent background. Under these conditions, the R, value of the intermediate toluene derivative was 0.46 and that of the corresponding benzyl alcohol was 0.36.

Isolation-The fermentations were terminated by the addition of 130 ml. of N NaOH and each tank was extracted with methylene dichloride (2x 20 liters). The extracts were reduced under vacuum and combined. Further reduction in volume resulted in a yellow crystalline precipitate weighing 150 g. Thin layer chromatography of this material showed traces of 2-chloro4-(1-piperazinyl) toluene with the major component chromatographically identical with a sample of 2 chloro 4 l-piperazinyl) benzyl alcohol prepared by a different method. (Refer to Example 4A or 7D below.)

Purification-The material was recrystallized from ethyl acetate (about 800 ml.). An insoluble fraction (4.5 g.) was filtered off; this material was probably a salt of the product since in the above basic TLC system it behaved like 2 chloro 4 (1 piperazinyDbenzyl alcohol. On cooling the filtrate in an ice bath, 120 g. of creamcolored crystalline 2 chloro 4 (l-piperazinyDbenzyl alcohol, MJP. 120-122 C. (corn), was obtained. A second crop of 23.5 g. was obtained from the mother liquor.

Analysis.--Calcd. for C H ClN O (percent): N, 12.36; C1., 15.64. Found (percent): N, 12.43; Cl, 15.44.

Infrared, ultraviolet and nuclear magnetic resonance spectral data of the above product show it to be identical with the product obtained hereinbelow (Example 4A or 7D) by reduction of the corresponding 2 chloro 4 (1- piperazinyl)benzaldehyde.

EXAMPLE 2 Fermentative enzymatic oxidation of 2-chloro-4-(2- diethylaminoethylamino)toluene to 2 chloro 4 (2- diethylaminoethylamino)benzyl alcohol was carried out following the procedure described in Example 1, using one 10-liter fermenter, Aspergillus sclerotiorum (SWRI A a sterile soy-dextrose medium of the following composition.

Tap water, 9.5 liters.

pH of about 7 prior to autoclaving at 120 C. and 15 lbs.

p.s.i. for 15 minutes,

161 g. of 2-chloro-4-(2-diethylaminoethylamino)toluene hydrochloride added in 3-15 g. portions over a period of eight days, a conversion temperature of 29-30 C., an aeration rate of 5 liters/minute, an agitation rate of 450 r.p.m., 150 ml. of 10 N sodium hydroxide solution to terminate the fermentation, and two liter portions of methylene dichloride to extract the product. A TLC on the extract showed good conversion to the corresponding benzyl alcohol, along with some of the corresponding 2 chloro 4 (2 diethylaminoethylamino)benzaldehyde, as shown by spraying the TLC plate with 2,4-dinitrophenylhydrazine. The methylene dichloride extract was concentrated in vacuo to an oil residue which was taken up with 200 ml. of anhydrous" methanol. To this solution was added sodium borohydride in 2 g. portions until a TLC showed no more aldehyde to be present (total of 6 g. of NaBH, added) and a corresponding increase in the amount of the corresponding benzyl alcohol. The methanol was removed in vacuo and the residue taken up in 500 ml. of chloroform. This solution was washed several times with distilled water and the chloroform removed in vacuo. The remaining crystalline material was washed with n-hexane to yield 93 g. of the white crystalline product, 2 chloro 4 (Z-diethylaminoethylamino) benzyl alcohol, M.P. 66.0-67.4 C. (corn). A second crop weighing 13.5 g. was obtained from the mother liquor.

Analysis.Calcd. for C H ClN O: N, Found: N, 11.09%.

Infrared spectral data of the above product show it to be identical with the product obtained by chemical methods as described in said US. Pat. 3,379,620, e.g., by reduction of ethyl 2-chloro-4-(Z-diethylaminothylamino) benzoate (Example 4A) and by reduction of ethyl 2- chloro 4(diethylaminoacetamido)benzoate (Example 6A).

Following the above-described screening procedure using Aspergillus sclerotiorum (SWRI A24), 2-chloro-4- (4-propionyl-l-piperazinyl)benzyl alcohol was prepared from 2 chloro 4 (4 propionyl 1 piperazinyl)- toluene as its p-toluenesulfonate acid-addition salt, which was prepared as follows: To 21.0 g. of l-(3-chloro-4- methylphenyl)piperazine and 50 cc. of pyridine was added, with cooling, 14.5 g. of propionic anhydride. The resulting mixture was allowed to stand at room temperature for three days, warmed on a steam bath, and then poured into water. The mixture was stirred for one hour and then extracted with ether. The ether extract was washed successively with aqueous sodium bicarbonate solution and water, dried over magnesium sulfate monohydrate, and concentrated to yield 26 g.-of a viscous oily material. The oil was distilled to yield 21.5 g. of 2-chloro- 4-propionyl-1-piperazinyl)toluene. B.P. 175-183 C. at 0.1-0.2 mm. This product was treated with 17 g. of ptoluenesulfonic acid monohydrate in l00'cc. of warm absolute ethanol. The resulting precipitate was collected, washed with cold absolute ethanol, and dried overnight at C. and 20 mm. to yield 29.5 g. of 2-chloro-4-(4- propionyl-1-piperazinyl)toluene p-toluenesulfonate, M.P. 194.0-195.2 C. (corn).

Analysis.-Calcd. for C14H19C1N2O'C7H3O3S (percent): Cl, 8.08; N, 6.38. Found (percent): Cl, 8.35; N, 6.07.

EXAMPLE 3 G. Dextrose 200 Soybean meal 200 NaCl 50 Yeast 50 K HPO 50 Tap water, 10 liters.

pH adjusted to 7.0 with 10 N HCl prior to autoclaving,

and 25 g. of 2 chloro 4 (4 thiocarbamyl 1 piperazinyl)toluene per fermenter. The inoculum (7%) was prepared from agar slant and growth in 700 ml. of the above medium for seventy-two hours at 26 C. on a rotary shaker at 410 r.p.m. The three l0-liter fermentations were carried out at 28 C. with an air flow of liters per minute and were agitated at 450 r.p.m. After an initial twenty-five hours growth, 5 g. portions of 2 chloro 4 (4 thiocarbamyl-1-piperazinyl)toluene hydrochloride, each dissolved in 5 ml. of dimethyl formamide, were added over a period of three days for a total of 25 g. in each tank. After seventy-two hours, the contents of each tank were made alkaline with concentrated ammonium hydroxide and extracted with 20 liters of methylene dichloride. The extracts were concentrated in vacuo and the residues combined. The resulting dark brown oil was mixed with 40 g. of silica gel and placed on a 300 g. silica gel column (3.5 x 45 cm.). The column was developed with increasing amounts of ether in n-hexane containing 1% triethylamine and finally with increasing amounts of methanolin-ether containing 1% triethylamine. The latter fractions eluted with methanol were combined, evaporated and the residue crystallized from a methanolwater mixture to yield 6 g. of crystalline solid. The solid was recrystallized from 100 ml. of isoamyl alcohol to yield 2.7 g. of solid. Two hundred mg. of the solid was dissolved in water, the solution made alkaline with a few drops of 10 N sodium hydroxide solution and extracted with methylene dichloride. Evaporation of the methylene dichloride in vacuo and drying the crystalline residue at 90 C. for ten hours yielded 130 mg. of 2-chloro-4-(1- piperazinyl)benzyl alcohol, whose infrared spectrum was found to be identical with the same product obtained hereinbelow in Example 4. By treating this compound in aqueous hydrochloric acid solution with a molar equivalent quantity of ammonium thiocyanate in water, warming the resulting solution, cooling the reaction mixture, and collecting the precipitate, there is obtained 2-chloro-4-(4-thiocarbamyl-1-piperazinyl)benzyl alcohol.

EXAMPLE 4 (A) 2 chloro 4 (1 piperazinyl)benzyl alcohol- A solution of 4 g. of 1-(3-chloro-4-carbethoxyphenyl) piperazine in ml. of anhydrous ether was added to a slurry of 600 mg. of lithium aluminum hydride in 500 ml. of ether at 0 C. The vigorously stirred reaction was treated with an excess of 35% aqueous sodium hydroxide solution after one hour; the layers were separated; and the aqueous layer was extracted with ether. The ether extract was dried over anhydrous magnesium sulfate and concentrated to a waxy solid. The solid was recrystallized once from ethyl acetate-n-pentane and once from isopropyl acetate to yield 1.4 g. of 2-chloro-4-(l-piperazinyl) benzyl alcohol, M.P. 121.4-123.2 C. (corr.).

AnaIysis.Calcd. for C H ClN O (percent): N, 12.36; C], 15.64. Found (percent): N, 12.15; Cl. 15.87.

2 chloro 4 (1 piperazinyl)benzyl alcohol hydrochloride-To 1 g. of 2-chloro-4-(l-piperazinyl)benzyl alcohol dissolved in acetone was added an excess of 9 N HCl in ethanol. The precipitate was collected, recrystallized from ethanol-acetone, and dried at 95 C. for twelve hours to yield 1.2 g. of the white crystalline 2- chloro-4-( lpiperazinyl)benzyl alcohol hydrochloride, M.P. 157.0159.0 C. (corr.) with decomposition.

Analysis.-Calcd. for C H ClN O-HCl (percent): C], 26.94; N, 10.64. Found (percent): Cl, 26.72; N, 10.52.

(B) The above intermediate 1-(4-carboethoxy-3-chlorophenyl)piperazine was prepared as follows: A solution of 24 g. of ethyl 4-amino-2-chlorobenzoate and 34 g. of bis(2-bromoethyl)amine hydrobromide in 500 ml. of nbutanol was refluxed for one hour and after standing overnight was treated with 13 g. of anhydrous sodium carbonate. After thirty hours of refluxing, the n-butanol was removed under reduced pressure leaving a waxy solid. The material was freed from inorganic salts by digesting it in absolute ethanol, filtering the mixture through infusorial earth, and adding ether to the filtrate to reprecipitate the product; after two such treatments, the product as the hydrobromide salt weighed 10 g. The free base was extracted from a basic solution with ether and 10 distilled, B.P. 165-175 C./0.05 mm. A picrate was prepared and recrystallized from ethanol, M.P. 235-237 C. Analysis.Calcd. for C H ClN O -C H N O N, 14.06%. Found (percent): N, 13.53%.

EXAMPLE 5 (A) 2-chloro-4-(4-ethyl-1-piperazinyl)benzyl alcohol from 2 chloro-4-(4-ethyl-l-piperazinyl)benzaldehyde.- To a solution containing 4.0 g. of 2-chloro-4-(4-ethyl-1- piperazinyl)benzaldehyde (see Example 5 (B)) in 45 ml. of absolute ethanol was added 10 ml. of a 50% solution of sodium borohydride in ethanol. The resulting mixture was stirred at room temperature for one hour and then allowed to stand over the weekend. After removal of the alcohol by distillation in vacuo, 20 ml. of 20% aqueous sodium hydroxide solution was added and the resulting mixture was extracted three times with ether. The ether extract was dried over anhydrous magnesium sulfate and the ether removed to yield an oil which solidified on cooling. The solid was recrystallized twice from benzene-n-hexane, using decolorizing charcoal the second time, and dried at C. for three hours at 20 mm. to yield 1.8 g. of 2-chloro-4-(4-ethyl-1-piperazinyl)benzyl alcohol, M.P. 96.8-99.0 C. (corr.).

Analysis.Calcd. for C H CIN O (percent): Cl, 13.91; N, 11.00. Found (percent): Cl, 13.78; N, 11.05.

(B) The above intermediate 2-chloro-4-(4-ethyl-1- piperazinyl)benzaldehyde was prepared as follows: To a solution under an atmosphere of nitrogen and containing 10.0 g. of sodium S-nitro-o-toluenesulfonate, 1.6 g. of ammonium chloride and 40 ml. of distilled water was added 8 g. of zinc dust in two portions, while cooling the reaction mixture to keep the temperature below 70 C. The excess zinc dust was filtered off and washed with 25 ml. of water; 2.4 ml. of concentrated hydrochloric acid was added to the filtrate. The resulting solution was added to a solution obtained by dissolving 6.0 g. of 1-(3-chlorophenyl)-4-ethylpiperazine in a mixture of 4.0 ml. of concentrated hydrochloric acid and 0.5 ml. of water followed by addition of 2.3 ml. of 37% aqueous formaldehyde, the first solution being added to the second immediately after addition of the formaldehyde to form the second solution. The reaction mixture was then allowed to stand at room temperature for two days, 50 ml. of a saturated solution of sodium chloride in water was added, and the resulting mixture was neutralized to a pH of 6.8-7.0 with ammonium hydroxide, whereupon a gummy yellow precipitate separated. The residue was stirred in an ice bath for thirty minutes and the resulting gummy precipitate was collected. To the precipitate was added 50 ml. of water and 25 ml. of ammonium hydroxide, the mixture was heated below its boiling point for ten minutes, cooled and extracted four times with ml. of ether. The ether extract was dried over anhydrous magnesium sulfate and the ether removed to yield, as a viscous yellow oil, 4.0 g. of 2-chloro- 4-(4-ethyl-l-piperazinyl)benzaldehyde. Infrared spectral analysis of this compound showed it to have a carbonyl group. Also, it was converted into its thiosemicarbazone, M.P. 223.0-223.5 C. (corn) with decomposition.

Analysis.Calcd. for C H ClN S (percent): 10.88; S, 9.84. Found (percent); Cl, 11.18; S, 9.46.

(C) The intermediate 1-(3-chlorophenyl)-4-ethylpiperazine used in Example 5(B) was prepared as follows: A mixture containing 15 g. of l-(3-chlorophenyl)pipera- Zine, 6 ml. of ethyl bromide and 100 ml. of benzene was refluxed for three hours. The white solid [l-(3-chlorophenyl)piperazine hydrobromide] that separated was filtered off, 15 ml. of ethyl bromide was added to the filtrate and refluxing was continued for another four hours. The mixture was stirred at room temperature overnight and another 3.5 g. of l-(3-chlorophenyl)piperazine hydrobromide was filtered off. The filtrate was concentrated in vacuo and the remaining oil was dissolved in anhydrous ether and filtered through infusorial earth (Super- Cel). To the filtrate was added 7.9 ml. of 9.7 N ethanolic hydrogen chloride. The resulting white precipitate was collected, air-dried and recrystallized from about 400 ml. of acetone to yield 5.0 g. of l-(3-chlorophenyl)-4-ethylpiperazine hydrochloride, M.P. 206208 C., after drying at 70 C. at 20 mm. for one hour. A second crop of 1.5 g., M.P. 2-08-209 C., was obtained by concentrating the filtrate to 100 ml. For analysis, a 0.5 g. sample of the first crop was placed in an oven at 7 C. and 20 mm. overnight.

Analysis.CalCd. for C H CIN HCI: N, 10.72%. Found: N, 10.78%.

EXAMPLE 6 (A) 2-chloro-4-(4-methyl-l-piperazinyDbenzyl alcohol from 2-chloro-4-(4-methyl-l-piperazinyl)benzaldehyde. To a solution of 12 g. of 2-chloro-4-(4-rnethyl-l-piperazinyl)benzaldehyde (Ex. 6(B)) in 100 ml. of methanol was added 50 ml. of water. The resulting cloudy solution was cooled and to it was added 2.0 g. of sodium borohydride in 20 ml. of water, followed by an additional 1 g. of sodium borohydride in 10 ml. of water. The resulting mixture was allowed to stand overnight. Most of the methanol was then removed by distilling in vacuo; 15 ml. of 35% aqueous sodium hydroxide was added to the aqueous mixture; and the alkaline solution was extracted three times with ether. The extracts were dried over anhydrous magnesium sulfate and concentrated in vacuo to yield an oily material which solidified on stand ing. The solid was recrystallized once from benzene-npentane and once from benzene to yield 4.5 g. of 2-chloro- 4-(4-methyl-1-piperazinyl)benzyl alcohol, M.P. 119.4- 12l.6 C. (corr.) when dried at 60 C. and 20 mm. for four hours. The structure of the product was confirmed by its infrared spectrum and the following analysis.

Analysis.Calcd. for C H ClN O (percent): C], 14.72; N, 11.63. Found (percent): CI, 14.72; N, 11.82.

(B) The above intermediate 2-chloro-4-(4-methyl-1- piperazinyl)benzaldehyde was prepared following the procedure described in Example 5(B) using 29.0 g. of sodium S-nitro-o-toluenesulfonate, 4.4 g. of ammonium chloride, 115 ml. of distilled water, 23 g. of zinc dust (90%), 70 ml. of wash water and 10.5 ml. of concentrated hydrochloric acid to form the first solution; and, 1 8.5 g. of 1-(3-chlorophenyl)-4-methylpiperazinyl in a mixture of 11.6 ml. of concentrated hydrochloric acid and 1 ml. of water, followed by addition of 6.4 ml. of 37% aqueous formaldehyde to form the second solution. The solutions were mixed and the reaction mixture worked-up as in Example 7B. There was obtained, as an oil, 15 g. of 2-chloro-4-(4-methyl-l-piperazinyl)benzaldehyde. Its IR spectrum in chloroform showed a carbonyl group; and, its semicarbazone melted at 231-233 C. and ana lyzed as follows.

Analysis.--Calcd. for C H ClN O' (percent): 1199; N, 23.68. Found (percent): CI, 12.32; N, 24.28.

(C) The intermediate 1-(3-chlorophenyl)-4-methylpiperazine used in Example 6(B) was prepared as follows: T o a solution containing a 43 g. portion of 1-(3-chlorophenyl)piperazine in 40 ml. of formic acid was added 18 ml. of 37% formaldehyde solution. The reaction mixture was refluxed for five hours, cooled, treated with 18 ml. of concentrated hydrochloric acid and concentrated in vacuo. Water was added and the solution was made basic with aqueous potassium hydroxide solution. The alkaline mixture was extracted with methylene dichloride, the extracts dried over anhydrous magnesium sulfate, the methylene dichloride evaporated off, and the oily residue distilled to yield an 18.5 g. fraction of 1-(3- chlorophenyl)-4methylpiperazine boiling at 115-125 C. at 0.3 mm.

12 EXAMPLE 7 (A) 2-chloro-4-(4-fonnyl 1 piperazinyl)benzaldehyde.To ml. of dirnethylformamide was added dropwise 51.7 g. of phosphorous oxychloride. After the exothermic reaction had subsided, the solution was cooled and 86.0 g. of 1-(3-chlorophenyl)-4-formylpiperazine (Ex. 7(B)) was added. The reaction mixture was stirred on a steam bath for two hours, cooled and poured onto ice. Sodium hydroxide was added to adjust the pH to about 4 and then a saturated sodium acetate solution was added to a pH of about 6. The mixture was extracted with chloroform, the chloroform extract concentrated in vacuo, and 500 ml. of hot isopropyl acetate was added to the viscous residue. The hot supernatant liquid was decanted from a small amount of gummy residue, treated with decolorizing charcoal, filtered, and allowed to cool. The resulting precipitate was collected to yield 33 g. of 2- chloro-4-(4-formyl 1 piperazinyl)benzaldehyde, M.P. 122 C. A second crop of 20 g. of this product was obtained by concentrating the filtrate. 2-chloro-4-(4- formyl-1-piperazinyl)benzaldehyde is also obtained following the above procedure but using in place of dimethylformamide a molar equivalent quantity of diethylformamide, N-methyl-N-(unsubstituted-phenyl)formarnide or N- (4-methoxyphenyl)-N-methylformamide or using a molar equivalent quantity of phosphorous oxybromide in place of phosphorous oxychloride.

(B) The above intermediate 1-(3-chlorophenyl)-4- formylpiperazine was prepared as follows: A mixture containing 78.6 g. of 1-(3-chlorophenyl)piperazine, 22.2 g. of formic acid and 100 ml. of benzene was refluxed for two hours with a water-separator connected to the reaction vessel; 9 ml. of water was collected. The reaction mixture was concentrated in vacuo and the residue distilled under reduced pressure to yield 86.0 g. of 1-(3- chlorophenyl)-4-formylpiperazine, distilling at 153-164 C. at 0.1 mm.

(C) 2-chloro-4-(l-piperazinyl)benzaldehyde.-A mixture containing 53 g. of 2-chloro-4-(4-formyl-1-piperazinyl)benzaldehyde and ml. of 7% aqueous hydrochloric acid solution was stirred on a steam bath for fifteen minutes and cooled. The precipitate was collected and washed with acetone to yield 39.9 g. of 2-chloro-4- (l-piperazinyl)benzaldehyde as its hydrochloride, M.P. 260 C. with decomposition. 1A 10 g. sample was recrystallized from water-methanol using decolorizing charcoal and found to melt at 257.0-258.0 C. (corn) with decomposition.

Analysis.-Calcd. for C H ClN O.HCl (percent): Cl, 27.15; N, 10.73. Found (percent): Cl, 27.35; N, 1080.

2-bromo-4-( l-piperazinyl)benzaldehyde, 2 iodo-4-(lpiperazinyl)benzaldehyde or 2-fluoro-4-(1-piperazinyl) benzaldehyde is obtained by following the procedures of Examples 7(B), 7(A) and 7(C), respectively, starting with the appropriate 1-(3-hal0phenyl)piperazine in Example 7(B).

(D) 2-chloro-4-(l-piperazinyDbenzyl alcohol.To a solution containing 28.5 g. of 2-chloro-4-(l-piperazinyl) benzaldehyde hydrochloride in 200 ml. of methanol was added 8 ml. of 35% sodium hydroxide solution, and the mixture cooled to 0 C. To this cooled solution was added a solution of 4.5 g. of sodium borohydride in 40 ml. of water and the resulting mixture stirred in an ice bath for thirty minutes (initial temperature rose to 25 C.). The methanol was removed by distilling in vacuo on a steam bath and the residue was heated with 50 ml. of water, cooled and then extracted with chloroform. The extract was dried over anhydrous calcium sulfate, treated with decolorizing charcoal, filtered, and concentrated in vacuo to remove the chloroform. There was thus obtained 23.2 g. of 2-ch1oro-4-(l-piperazinyDbenzyl alcohol, M.P. 118-120 C.

2-bromo-4-(1-piperazinyl)benzyl alcohol, 2-iodo-4-(1- piperazinyl)benzyl alcohol or 2-fiuoro-4-(1-piperazinyl) benzyl alcohol is obtained following the procedure of Example 7( D) using the appropriate 2-halo-4-(1-piperazinyl)benzaldehyde as its hydrochloride.

EXAMPLE 8 (A) 2-chloro-4-(4-benzoyl-1-piperazinyl)benzaldehyde was prepared following the procedure described in Example 7(A) using 4.1 ml. of phosphorous oxychloride, 50 ml. of dimethylformamide and 15.0 g. of 1-(3-chlorophenyl)-4-benzoylpiperazine, and a heating period of four hours. The product which separated when the reaction mixture was made alkaline with sodium hydroxide was collected and recrystallized from isopropyl alcohol to yield 7.9 g. of 2-chloro-4-(4-benzoyl-1-piperazinyl)benzaldehyde, M.P. 155-160 C.

(B) The above intermediate l-(3-chlorophenyl)-4- benzoylpiperazine was prepared as follows: To a solution containing 30.0 g. of 1-(3-chlorophenyl)piperazine in 200 ml. of water and 50 ml. of ethanol cooled to C. was added simultaneously, over a period of about thirty minutes at 0 to 3 C. with stirring, 32.2 g. of benzoyl chloride and 25 ml. of 25% sodium hydroxide solution. To the mixture was added 100 ml. of ethanol and the resulting solution was stirred at 0 C. for two hours and then placed in a refrigerator for three days. The reaction mixture was then extracted with ether and the ether extract washed three times with water. The ether extract was extracted with 7% hydrochloric acid solution; however, on solidification of the acidic extract no product separated. The ether solution was then dried over anhydrous calcium sulfate and concentrated in vacuo to remove the ether. The residue was heated on a steam bath under high vacuum (about 0.5 mm.) to remove a few ml. of ethyl benzoate. The residue was then recrystallized from ether, washed successively with cold ether and n-pentane to yield 36.6 g. of 1-(3-chlorophenyl)-4-benzoylpiperazine, M.P. 75-77 C.

Analysis.Calcd. for C H 'CIN O (percent): 11.79; N, 9.32. Found (percent): 01, 11.94; N. 9.33.

(C) 2-chloro-4-(l-piperazinyl)benzaldehyde was obtained following the procedure described in Example 7 (C) using 10 g. of 2-chloro-4-(4-benzoyl-1-piperazinyl)benzaldehyde, 25 ml. of concentrated hydrochloric acid, 25 ml. of ethanol and a reflux period of five hours. There was thus obtained 3.5 g. of the compound, M.P. 252 C. with decomposition.

EXAMPLE 9 (A) 2-chloro 4 {4-[6 (4 tertiary pentylphenoxy) hexyl]1-piperazinyl}benzyl alcohol was prepared following the procedure described in Example (A) using 15 g. of 2-chloro-4-{4-[6-(4-tertiary-pentylphenoxy)hexyl]-1- piperazinyl}benzaldehyde, 100 ml. of methanol and 2 g. of sodium borohydride. The product was isolated from the reaction mixture by successive chromatographic separations on silica gel columns (100 g. each time, 2.5 x 50 cm.), first using 3% isopropylamine in ether and then 2% isopropylamine in ether. After recrystallization from methanol, there was thus obtained 0.9 g. of 2-chloro-4-{4-[6- (4 tertiary amylphenoxy)hexyl] 1 piperazinyl}benzyl alcohol, M.P. 71.8-73.6 C. (corr.).

Analysis.Calcd. for C H ClN O (percent): C, 71.08; H, 8.74; N, 5.92. Found (percent): C, 70.96; H, 8.91; N, 6.21.

(B) The above intermediate 2-chloro-4-{4-[6-(4-tertiary-pentylphenoxy)hexyl] 1 piperazinyl}benzaldehyde was prepared as follows: To 12 m1. of dimethylformamide cooled to 5 C. was added dropwise 3.7 ml. of phosphorous oxychloride. To this solution was added slowly over a period of seventy-five minutes, keeping the temperature at -20 C., a solution of 17.6 g. of 1-(3-chlorophenyl)-4-[6-(4-tertiary-amylphenoxy)hexyl]piperazine in 12 ml. of dimethylformamide. The resulting thick paste was stirred at room temperature for two hours and fifteen minutes, and then heated on a steam bath with stirring for two and one-half hours. The hot reaction mixture was poured onto ice and the resulting solution neutralized with 10% sodium hydroxide solution. The alkaline mixture was extracted with ether; the extract was dried over anhydrous calcium chloride and concentrated in vacuo to remove the ether. There was thus obtained, as an oil, 15 g. of 2-chloro-4-{4- 6- (4-tertiary-pentylphenoxy) hexyl] -1- piperazinyl}benzaldehyde.

(C) The intermediate 1-(3-chlorophenyl)-4-[6-(4-tertiary-amylphenoxy)hexyl]piperazine used in Example 9(B) was prepared as follows: A mixture containing 49 g. of 1-(3-chlorophenyl)piperazine, 50.5 g. of triethylamine, 81.5 g. of 6-(4-tertiary-amylphenoxy)hexyl bromide and 500 ml. of toluene was refluxed for six hours and filtered to remove some precipitated triethylamine hydrobromide. About 500 ml. of ether was added to the reaction mixture to precipitate more triethylamine hydrobromide and the resulting mixture was filtered through infusorial earth (Super-Cel). The filtrate was heated in vacuo to remove the solvents and yield 106 g. of an oil. A 10 g. sample of the oil was dissolved in ether and treated with a solution of hydrogen chloride in ethanol. The gummy precipitate was collected, recrystallized from about 200 ml. OfglSOPI'OPYl acetate, dried at C. and 20 mm. to yield 9 g. of 1-(3-chlorophenyl)-4-[6-(4-tertiary-amylphenoxy)hexyl]piperazine hydrochloride, M.P. 119-121 C. The remainder of the above oil (96 g.) was passed through a 200 g. column of silica gel in a 2% solution of isopropyl amine in ether. The first fraction of 1 liter yielded 59 g. of colorless oil which was taken up in 500 ml. of ether and treated with 17.5 ml. of 7.6 N ethanolic hydrogen chloride. The precipitate was collected, dried at 70 C. and 20 mm. for five hours to yield an additional 62 g. of 1-(3-chlorophenyl)-4-[6-(4-tertiaryamylphenoxy)hexyl]piperazine hydrochloride, M.P. 116- 119 C.

(D) The intermediate 6-(4-tertiary-amylphenoxy)hexyl bromide used in Example 9(C) was prepared as follows: A solution of sodium 4-tertiary-amylphenoxide was prepared by mixing 75 g. of 4-tertiary-arnylphenol, 18 g. of sodium hydroxide and 600 ml. of isopropyl alcohol This solution was added over a period of one hour to a refluxing solution of 1,6-dibromohexane in 600 ml. of isopropyl alcohol. The resulting reaction mixture was refluxed for one hour, about half of the solvent was removed in vacuo, and the remaining reaction mixture was poured into water. The mixture was extracted with ether; the ether extract was washed successively with 5% sodium hydroxide solution and twice with water, and then dried over anhydrous calcium chloride. The residue was distilled in vacuo to yield 98.0 g. of 6-(4-tertiaryamylphenoxy) hexyl bromide, distilling at -112 C. at 0.05 mm., n =1.5190.

EXAMPLE 10 2-chloro-4-[4-(3 carboxy-Z-propenoyl)-1-piperazinyl] benzyl alcohol.--A solution of 2.5 g. of maleic anhydride in ml. of benzene was added with stirring to a solution of 2.6 g. of 2-chloro-4-(1-piperazinyl)benzyl alcohol in 500 ml. of benzene over a period of thirty minutes. A slight rise in temperature to 40 C. was noted. After one hour, the finely divided solid was collected and dried for two hours at 0.1 mm. and room temperature to yield 3.2 g. of 2-chloro-4-[4-(3-carboxy-2-propenoyl)-1-piperazinyl]benzyl alcohol, M.P. 145.0-146.0 C. (corr.).

Analysis.-Calcd. f0! C15H1qC1N204 (percent): C, 10.92; N, 8.63. Found (percent): C, 10.75; N, 8.38.

EXAMPLE 1 1 Fermentative enzymatic oxidation of 2-chloro-4-[4-(2- hydroxyethyl)-1-piperazinyl]toluene to 2-chloro-4-[4-(2- hydroxyethyl)-1-piperazinyl]benzyl alcohol was carried out following the procedure described in Example 2 using 4.0 g. of 2-chloro-4-[4-(2-hydroxyethyl)-1-piperazinyl] toluene and a reaction period of two days, all other reaction conditions being the same as in Example 2. The product was isolated as follows: The oily residue after evaporation of the methylene dichloride was mixed with 30 g. of silica gel and placed on a 150 g. silica gel column (3.7 x 21 cm.) and the column was developed, first using increasing amounts of ether in n-hexane, and then using increasing amounts of methanol in ether (0.5% to 8% methanol), all solvents containing 0.5% triethylamine. The fractions collected with 3%8% of methanol in ether were combined and evaporated to yield an oily residue. The residue was crystallized from ethyl acetate using decolorizing charcoal and then recrystallized from ethyl acetate to yield 2.28 g. of 2-chloro-4-[4-(2-hydroxyethyl)- l-piperazinynbenzyl alcohol, M.P. 99.0'1.6 C. (corr.), after drying in a vacuum oven at 50 C. for twelve hours.

Analysis.Calcd. for C H CIN O (percent): Cl, 13.10; N, 20.35. Found (percent): Cl, 13.44; N, 10.06.

EXAJVIPLE l2 2 chloro4-(4-acetyl-l-piperazinyl)benzyl alcoholTo a solution containing 6.0 g. of 2-chloro-4-(l-piperazinyl)- benzyl alcohol in 6 cc. of pyridine was added 4 cc. of acetic anhydride. The resulting reaction mixture was heated on a steam bath for ten minutes and then allowed to cool to room temperature. The resulting precipitate was collected, washed successively with n-hexane and ether, recrystallized from ethyl acetate using decolorizing charcoal, and dried in a vacuum oven at 70 C. for eight hours to yield 4.8 g. of 2-chloro-4-(4-acetyl-1-piperazinyl)benzyl alcohol, M.P. 147.0-151.0 C. (corn).

Analysis.-Calcd. for C13H1'7N2O2C1 (percent): Cl, 13.19; N, 10.43. Found (percent): Cl, 13.22; N, 10.12.

Infrared spectral data of the above compound showed the presence of N-acetyl rather than O-acetyl. Running the above reaction using double the quantity of acetic anhydride, there is obtained 2-ch1oro-4-(4-acetyl-l-piperazinyl)benzyl acetate.

EXAMPLE 13 Fermentative enzymatic oxidation of 2-chloro-4-(4-benzyl-l-piperazinyl)toluene to 2-chloro-4(4-benzyl-l-piperazinyl)benzy1 alcohol was carried out following the procedure described in Example 2 using 5.0 g. of 2-chloro-4- (4-benzyl-l-piperazinyl)toluene hydrochloride and a reaction period of forty hours, all other reaction conditions being the same as in Example 2. The product was isolated as follows: The residue after evaporation of the methylene dichloride was mixed with 30 g. of silica gel and placed on a 150 g. silica gel column (3.5 x 25 cm.) and the column was developed using increasing amounts of ether in n-hexane and finally with increasing amounts of methanol in ether, all solvents containing 0.5 triethylamine. The fractions collected with 60-90% ether in nhexane and 0.5 triethylamine were combined and evap orated to yield a residue which was crystallized twice from ethyl acetate and once from isopropyl acetate to yield 1.23 g. of 2-chloro-4-(4-benzyl-1-piperazinyl)benzyl alcohol, M.P. 107.0-110.8 C. (corn).

Analysis.Calcd. for C H CIN O (percent): 11.19; N, 8.84. Found (percent): Cl, 11.22; N, 8.49.

EXAMPLE l4 Fermentative enzymatic oxidation of 2-chloro-4-(4-allyl l-piperazinyl)toluene to 2-chlo'ro-4(4-allyl-l-piperazinyl) benzyl alcohol was carried out following the procedure described in Example 2 using 60 g. of 2-chloro-4- (4-allyl-1-piperazinyl toluene hydrochloride and a reaction period of forty hours. The product was isolated as in Example 13; the fractions collected with 55-75% ether in n-hexane and 0.5 triethylamine' were combined and evaporated to yield a residue which was crystallized from ethyl acetate using decolorizing charcoal to yield 3.0 g. of 2-chloro 4-(4-allyl-1-piperazinyl)benzyl alcohol, M.P. 91.0-93.0 C. (corn).

16 Analysis.Calcd. for C H CIN O (percent): Cl, 13.29; N, 10.50. Found (percent): Cl, 13.54; N, 10.22.

EXAMPLE 15 3 chloro-N-(2-diethylaminoethyl)-4-methoxymethyl- 2,6-dimethylaniline.When 2-chloro-4-(Z-diethylaminoethylamino)-3,5-dimethylbenzyl alcohol (152 g.) dissolved in ethanol (150 ml.) was mixed with p-toluenesulfonic acid monohydrate (128 g.) in ethanol (400 ml.), there separated a white crystalline precipitate of 2-chlor0- 4-(2-diethylaminoethylamino) 3,5-dimethylbenzyl alcohol di-(p-toluenesulfonate) When the latter was dissolved in boiling absolute methanol, the hot solution treated with decolorizing charcoal and filtered, and the'filtrate allowed to cool, there separated 3-chloro-N-(2-diethylaminoethyl) 4 methoxymethyl-2,6-dimethylaniline di-(ptoluenesulfonate) (119 g.), M.P. 153.0-155.8 C. (corn) with decomposition.

Analysis.Calcd. for C H ClN O'2C- H O S (percent): S, 9.97; Cl, 5.56. Found (percent): S, 10.21; Cl, 5.61.

3 chloro-N-(Z-diethylaminoethyi)-4-methoxymethyl- 2,6-dimethylaniline in free base form was obtained by dissolving its di-(p-toluenesulfonate) in water, making the aqueous solution alkaline with aqueous sodium hydroxide solution, extracting the free base form with methylene dichloride, removing the methylene dichloride, and drying in vacuo at 65 C.

Following the foregoing procedure using, in place of boiling methanol, boiling n-propanol, n-butanol or n-hexanol to dissolve the 2-chloro-4-(2-diethylaminoethylamino)-3,5-dimethylbenzyl alcohol di-(p-toluenesulfonate), there is obtained the following ethers first in the form of their di-(p-toluenesulfonates) and then in their free base forms: 3-chloro-N-(2-diethylaminoethyl)-2,6-dimethyl-4-n-propoxy-methylaniline, 4 n-butoxymethyl-3- chloro-N-(2-diethylaminoethyl)-2,6-dimethylaniline or 3- chloro N (Z-diethylaminoethyl)-4-n-hexoxymethyl-2,6- dimethylaniline, respectively. Similarly, following the above procedures using, in place of 2-chloro-4-(2-diethylaminoethylamino)-3,5-dimethylbenzyl alcohol, 2-chloro- 4-(2-diethylaminoethyl)benzyl alcohol, 2-chloro-4-(4allyl-l-piperazinyl)benzyl alcohol or 2-chloro-4-(4-methyl- 1-piperazinyl)benzyl alcohol, there is obtained first the respective benzyl alcohols as their di- (p-toluenesulfonates) and then, after treatment of the latter with boiling methanol, the following methyl ethers, first in the form of their di-(p-toluenesulfonates) and then in their free base forms: 3 chloro N-(Z-diethylaminoethyl)-4-methoxymethylaniline, 4-a11yl-1-(3-chloro-4-methoxymethylphenyl)piperazine or 1-(3-chloro-4-methoxymethylphenyl)-4- methylpiperazine, respectively.

EXAMPLE 16 Fermentative enzymatic oxidation of 2-chloro-3,5-dimethyl-4-(l-piperazinyl)toluene (6 g.) to 2-chloro-3,5-dimethyl-4-(1-piperazinyl)benzyl alcohol (0.825 g.) was carried out following the procedure described in Example 2 using a reaction period of seven days, a conversion temperature of 28-29 C., an agitation rate of 400 r.p.m. an aeration rate of 4 liters per minute, and 120 ml. of 10 N sodium hydroxide solution to terminate the fermentation. The product was isolated as follows: The oily residue after evaporation of the methylene dichloride extracts was taken up in 300 ml. of methanol and the resulting solution extracted with two ml. portions of n-hexane. Concentration of the methanol solution yielded 9 g. of viscous oil which was dissolved in 25 ml. of methylene dichloride and applied to 20 x 40 cm. thick layer (1 mm.) silica gel plates, twenty plates being used. The plates were developed in chloroform (87) :methanol (10): isopropylamine (3) (parts by volume). The bands corresponding to the more polar conversion product were removed from each plate and eluted with 300 ml. of methanol. The methanol solution was first filtered through fine filter paper (Whatman No. 1), concentrated in vacuo to about 25 ml. and filtered through an ultra-fine sintered glass disc. The remaining methanol was removed in vacuo to yield 1.5 g. of oily material which was crystallized from ethyl acetate to yield 0.825 g. of 2-chloro-3,5-dimethyl-4- (l-piperazinyl)benzyl alcohol, M.P. l44-l45 C.

Analysis.--Calcd. for C H ClN O (percent): Cl, 13.92; N, 10.99. Found (percent): Cl, 14.05; N, 10.76.

EXAMPLE 17 1-(3-chloro-4-methoxymethylphenyl)piperazine as its p-toluenesulfonate, 48.7 g., M.P. 164-165 C., was prepared by adding to a solution of 33 g. of 2-chloro-4-(1- piperazinyl)benzyl alcohol in 275 ml. of warm methanol a solution of 54.1 g. of p-toluenesulfonic acid monohydrate dissolved in 275 ml. of warm methanol, heating the reaction mixture to boiling, cooling the reaction mixture, collecting the crystalline product and recrystallizing it from methanol.

EXAMPLE l8 1-(3-chloro-4-isopropoxymethylphenyl)piperazine was obtained following the procedure described in Example 15 using boiling isopropyl alcohol instead of boiling methanol. Thus, from g. of 2-chloro-4-(l-piperazinyl) benzyl alcohol as its di-(p-toluenesulfonate) in 500 ml. of isopropyl alcohol and 8.4 g. of p-toluenesulfonic acid monohydrate in 200 ml. of isopropyl alcohol, there was obtained 12.8 g. of 1-(3-chloro-4 isopropoxymethylphenyl)piperazine p-toluenesulfonate, M.P. 156160 C.

EXAMPLE 20 4 (3 chloro-4-hydroxymethylphenyl)-a,a-dimethyl-lpiperazineethanol, same as 1-(3-chloro-4-hydroxymethylphenyl) 4 (2-hydroxy-2-methylpropyl)piperazine.To 22.6 g. of 2-chloro-4-(l-piperazinyl)benzyl alcohol in 150 ml. of ethanol was added 27 ml. of isobutylene oxide (prepared by reacting l-chloro-2-methyl-2-propanol with 10 N aqueous sodium hydroxide solution and recovering the upper layer); the resulting mixture was refluxed for three hours and allowed to cool. The reaction mixture was filtered and to the filtrate was added 18 ml. of 5.92 N ethanolic hydrogen chloride. The resulting white crystalline precipitate was collected and dried in vacuo over phosphorus pentoxide at 60 C. for eight hours and then at 40 C. for three days to yield 22 g. 4-(3-chloro-4-hydroxymethylphenyl)-u,-din1ethyl-l-piperazineethanol, as its hydrochloride, M.P. 189190 C.

EXAMPLE 21 4 (3-chloro-4-methoxymethylphenyl)-u,a-dimethyl-1- piperazineethanol.To a solution containing 10 g. of 4-(3- chloro 4 hydroxymethylphenyl)-a,a-dimethyl-1-piperazineethanol hydrochloride in 100 ml. of methanol was added a few drops of 6 N ethanolic hydrogen chloride and the resulting mixture was heated on a hot plate for one hour, concentrated to a volume of about 25 ml., removed from the hot plate, treated with ether to incipient cloudiness and allowed to stand. The resulting crystalline precipitate was collected and dried in vacuo over phosphorus pentoxide for eight hours at 60 C. to yield 4.2 g. 4-(3- chloro 4 methoxymethylphenyl)-u, 1-dimethyl-l-piperazineethanol as its hydrochloride, M.P. l25-l26 C.

18 EXAMPLE 22 4 (3-chl0ro-4-isopropoxymethylphenyl)-a,a-dimethyl- 1-piperazineethanol.-To a solution containing 10 g. of 4 (3 chloro-4-hydroxymethylphenyl)-a,a-dimethyl-l-piperazineethanol hydrochloride in hot isopropyl alcohol (about 1.5 l.) was added 0.5 ml. of 6 N ethanolic hydrogen chloride and the resulting solution was heated gently for thirty minutes, treated with anhydrous sodium sulfate and decolorizing charcoal and filtered. The filtrate was concentrated in vacuo to about 200 ml. and chilled in an ice bath. The resulting white crystalline precipitate was collected and dried in vacuo over phosphorus pentoxide for ten hours to yield 9.35 g. of 4-(3-chloro-4-isopropoxymethylphenyl)-a,m-dimethyl-l-piperazineethanol as its hydrochloride, M.P. 178l79 C.

EXAMPLE 23 4 (3 chloro-4-ethoxymethylphenyl)-u,m-dimethyl-1- piperazineethanol as its hydrochloride, 7.0 g., M.P. 124- 125 C. was prepared as in Example 22 using 10 g. of 4-(3-chloro-4-hydroxymethylphenyl)-a,u-dimethyl-l-piperazineethanol hydrochloride, 1.5 l. of ethanol, 0.3 ml. of 6 N ethanolic hydrogen chloride and a reflux period of one hour.

Following the procedure described in Example 1 using the appropriate 4-[amino-(polycarbon-lower-alkyl)-amino]-2-halotoluene, the corresponding 4-[amino-(po1ycarbon-lower-alkyl)-amino]-2-halobenzy1 alcohols are obtained, as illustrated, without limiting the foregoing, by the compounds of Table A.

TABLE A ('lHaoH.

-hal

| $Hg CH3 CE: /CH:

Hal R4 a s H CH3 CH3 H H H H H H H H H CHIC (CH3)=CH H H CONHI H H COOC2H5 CH3 H CH7CH2OH C2115 CzHg (GH2)4OH H H coon: H H OOCZHTH H H CO(CH2)ZCOOH H H (I311; (3H H H CmnmQI J-CHrc-Om CH3 CH;

14 Br H H (oHmo- C(CH CH;CH

15.... 01 01 H H mum-@omormom 16.... 01 COOC3H1-n H H 17 01 COCHQCH; H H 18 c1 CflHu-n H H 19..-- c1 CHQCH3C6H5 H H (oH, .o -o elm-n The 4- [amino- (polycarbon-lower-alkyl -amino] -2-halobenzyl alcohols of Table A also can be prepared by following the procedure described in Example 4(A) using a corresponding molar equivalent quantity of the appropriate ethyl or other lower-alkyl 4-[amino-(polycarbonlower-alkyl)amino]-2-halobenzoate or by following the procedure described in Examples 5 (A), 6(A), 8(D) or 9(A) using a corresponding molar equivalent quantity of the appropriate 4 [amino (polycarbon lower alkyl)- amino]-2-halobenzaldehyde; the latter benzaldehydes can be obtained by following the procedure described in Examples 7(A), 8(A) or 9(B) using corresponding molar equivalent quantities of the appropriate 3-(1-piperazinyl) halobenzene, dimethylformamide and phosphorus oxychloride or by following the procedure described in Examples 5 (B) or 6(B) using the appropriate intermediates prepared as illustrated in Examples 5(0) or 6(C).

The 4 [amino (polycarbon lower-alkyl)-amino]-2- halobenzyl alcohols and lower-alkyl ethers of our invention when administered orally to hamsters and Swiss mice infected with Schistosoma mansoni were found to clear completely the animals of the parasitic infection at varying dose levels of compound per kg. of body weight per day for five consecutive days. Some of the most active embodiments have ED values below 20 mg. per kg. of body weight, ED meaning the effective dose necessary to clear 50% of the hamsters or mice (at least 5 animals at each of three or more dose levels) of the amebic infection. Generally, our 4-[amino-(polycarbon-lower-alkyl)- amino]-2-halobenzyl alcohols and ethers have greater activity in hamsters and have greater or approximately the same activity in mice when compared with the corresponding heretofore known 4-[amino-(polycarbon-loweralkyl)-amino]-2-hal0toluenes. Also, our benzyl alcohols were generally less toxic than the corresponding known toluene derivatives when determined by standard procedures in Swiss mice. Illustrative of the schistosomacidal activity of our compounds when determined as described above are the following data: 2-chloro-4-(Z-diethylaminoethylamino)benzyl alcohol, oral ED values of 9.0124 mg./kg./day and 15.0-12.8 mg./kg./day in hamsters and Swiss mice, respectively, compared with respective values of 45116.6 mg./kg./day and 13.0127 mg./kg./day for the corresponding toluene derivative, i.e., 2-chloro-4-(2- diethylaminoethylamino)toluene (HCl salt); 2-chloro-4- (l-piperazinyl)benzyl alcohol, oral ED values of about 12.5 mg./kg./day and 2.1105 mg./kg./day in hamsters and Swiss mice, respectively, compared with respective values of 84129.3 mg./kg./day and 7.5120 mg./kg./ day for the corresponding toluene compound (di-HCl salt); 2-chloro-4-(4-ethyl-1-piperazinyl)benzyl alcohol, oral ED values of 175181 mg./kg./day and 4111.8 mg./kg./day in hamsters in Swiss mice respectively; 2- chloro 4 [4 (3 carboxy 2 propenoyl) 1 piperazinyl]benzyl alcohol, oral ED of 6.0113 mg./kg./day in mice; 2-chloro-4-(2-diethylaminoethylamino)-3,5-dimethylbenzyl alcohol di-(p-toluenesulfonate), oral ED of 3712.0 mg./kg./day in Swiss mice; 3-chloro-N-(2-diethylaminoethyl) -4-methoxymethyl-2,6-dimethylaniline di- (p-toluenesulfonate), oral ED of 3.410.5 mg./kg./day in Swiss mice; 3-chloro-4-ethoxymethyl-N-(Z-diethylaminoethyl) -2,6-dimethylaniline di- (p-toluenesulfonate) oral ED of about 12.5 mg./kg./day in hamsters. Illustrative of the toxicity of our compounds are the following acute intravenous LD values in Swiss mice, LD meaning the dose lethal to 50% 0f the mice (10 mice tested at each of three dose levels): 2-chloro-4-(2-diethylaminoethylamino)benzyl alcohol, LD of 5112 mg./kg., compared with 3312.2 mg./kg. for the corresponding 2- 20 chloro-4-(2-diethylaminoethylamino)-toluene; 2 chloro- 4-(l-piperazinyl)benzyl alcohol, LD of 5214 mg./kg., compared with 3912.6 mg./kg. for the corresponding toluene compound.

The subject matter which the applicants regard as their invention is particularly pointed out and distinctly claimed as follows:

1. A member selected from the group consisting of a compound of the formula:

where ha! is halo; R R and R are each hydrogen or lower-alkyl; NB is CH2CH2 NR4 CH: 0112 and R is hydrogen, lower-alkyl, lower-alkanoyl having from one to six carbon atoms, lower-carbalkoxy where alkoxy has from one to six carbon atoms, carboxy-(loweralkanoyl) Where lower-alkanoyl has from one to six carbon atoms, carboxy-(lower-alkenoyl) where lower-alkenoyl has from three to six carbon atoms or phenyl-X- (lower-alkyl) where X is oxygen or a direct linkage and phenyl is unsubstituted or substituted by from one to two substituents selected from alkyl having from one to eight carbon atoms, halo, lower-alkenyl having from three to six carbon atoms and lower-alkoxy having from one to six carbon atoms, lower-alkyl in each instance having from one to six carbon atoms and being primary or secondary; and chemotherapeutically acceptable salts thereof.

2. 4-piperazino-2-halobenzyl alcohol according to claim 1 where R is hydrogen and NB is piperazino.

3. 2-chloro-4-(l-piperazinyl)benzyl alcohol according to claim 1.

4. 2 chloro 4 [4 (3 carboxy 2 propenoyl) 1- piperazinyl]benzyl alcohol according to claim 1.

5. 2-chloro-4-(4-methyl-1-piperazinyl)benzyl alcohol according to claim 1.

6. 2-chloro-4-(4-ethyl-1-piperazinyl)-benzyl alcohol according to claim 1.

7. 2 chloro 4 [4 (2 hydroxyethyl) 1 piperazinyl]benzyl alcohol according to claim 1.

8. 2 chloro 4 {4 [6 (4 tertiary amylphenoxy) hexyl]-1-piperazinyl}benzyl alcohol according to claim 1.

9. 2 chloro 4 (4 benzyl 1 piperazinyl)benzyl alcohol according to claim 1.

10. 2-chloro-4-(4-allyl-l-piperazinyl)benzyl alcohol according to claim 1.

11. 2 chloro 3,5 dimethyl 4 (l piperazinyl) benzyl alcohol according to claim 1.

References Cited UNITED STATES PATENTS 2,875,205 2/ 1959 Ruschig et al 260268 C 2,945,860 7/1960 Schmidt-Barbe 260168 C DONALD G. DAUS, Primary Examiner U.S. Cl. X.R. 260268 PH lnvcmzm-(s) Sydney Archer and David Rosi It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

i Column 3, line 19, 'benzene ring or phenyl can bear .7

any member" should read benzene ring of phenyl can bear any number I Column 3, line 45, omit line 45 i.e., -CH CH CHCH Column 4, line 37, between "fonate, and "quinate, insert benzenesulfonate, f

Column 4, line "acid-solution" Should read acid-addition Signed and sealed this 20th day of November 1973;

(SEAL) Attest: V EDWARD M.FLETCHE R ,J R.' De TEGTMEYEF V Attesting officer Acting commissioner of Patents 

